Human and animal tissues have been studied, at great length, and numerous defects have been identified at the cellular level. Many of these defects have a genetic basis and are due, in many cases, to a defective or missing gene. The defect may be the result of a point mutation or other cause, leading to a disruption or abnormal change in the nucleotide sequence within the gene itself. The ultimate result of a malfunctioning gene is, of course, the failure to produce gene protein product or, alternatively, the production of gene protein product which is itself defective.
In the event of the identification of a defective gene, in the human or in an animal, gene therapy may be performed. In this regard, a cloned gene may be delivered to the nucleus of the cell to be treated for the purpose of rectifying the abnormal or defective genetic material. The material utilized in this process is expensive to produce, requiring sophisticated laboratory equipment and the practice of sophisticated molecular genetic techniques. Such techniques are not generally available and since they are, for the most part, confined to a relatively small number of highly sophisticated molecular genetic laboratories.
Because of the cost and general unavailability of gene therapeutic methods, alternative forms of cell therapy are desirable. Analysis of the molecular structure and function of the protein product enables conclusions to be drawn as to the health of the gene producing the product. Frequently, gene protein products can be used for evaluation of interaction among genes for, as an example, the determination of the tumor suppression mechanisms of the body. Regarding tumor suppression mechanisms, reference may be made to the foregoing parent patent applications. In order to facilitate the elucidation of gene function and interaction, it would be highly desirable to have methods for producing gene protein products which were reliable, inexpensive, and which could provide large volumes of the protein in a reliable and predictable manner.
In addition to the value of the gene protein product in the elucidation of genetic function and interaction, the protein itself can be used therapeutically for treatment of defective genetic conditions. In such cases, it would be convenient and effective to introduce into a cell, having defective genetic material, the appropriate gene protein product. Delivery of the protein product would, in some cases, be less expensive and more easily accomplished than the therapeutic administration of the genetic material itself.
For protein therapy, reference may be made to the foregoing mentioned related patent application filed contemporaneously herewith.
As a result of an appreciation of the importance of gene protein products, it would be highly advantageous to have available a technique for preparing and isolating gene protein products in substantially purified form. The availability of intact and biochemically active protein in large quantities, would represent a significant advance for studying the biochemical properties and molecular behavior involved in genetic mechanisms, as well as for therapeutic applications.
In general, for laboratory purposes as distinct from large scale production, gene protein products have been procured from cells, as well as by synthetic production thereof. With regard to derivation from cells, cellular proteins exist only in very small quantities. As a result, it is not practical to attempt to derive sufficiently large quantities of the protein from natural sources.
With regard to synthetic methods of production, attempts to express protein by introducing the coding sequence of a gene into a bacterial expression vector, have only been partially successful. Bacterially produced proteins have poor solubility. Another drawback of using a bacterial expression system is that bacterial cells are unable to modify eukaryotic proteins, and analysis of such proteins could be misleading, if post-translational modifications are required for the normal function of the protein. In summary, bacterially produced proteins generally have poor solubility and may be molecularly defective, thereby limiting their value.
Conventional laboratory techniques for making protein products have suffered from an inability to produce sufficiently large quantities, but also the resulting products have not been sufficiently pure, on a consistent basis. As a representative example of the difficulty in the production of some protein products, TrpE-RB fusion proteins have been developed and a T7 RNA polymerase expression has been utilized, expressing in E.coli, for production of the polypeptide. These methods have proven to be relatively complicated, requiring the practice of sophisticated biochemical techniques. In addition, such methods have serious limitations, since they are capable of producing only very small amounts of the desired polypeptide. In addition, the polypeptides produced by such methods are often not molecularly suitable, as for example, not being phosphorylated.
Therefore, in view of the importance of the gene product polypeptides, it would be highly desirable to have a method for producing such polypeptides, in substantial quantities, having desired biochemical and biophysical characteristics.